CD4+ T cells play important roles in adaptive immunity as mediators of inflammation, tolerance, and as facilitators of B cell maturation (Pao et al., 1996; Klein et al, 2010). The human CD4+ T cell repertoire contains millions of different cells that vary in the T cell receptors (TCR) they express. Indeed, many human disorders involve discrete subsets of CD4+ T cells that proliferate in response to highly specific antigens, and the isolation of the small proportion of CD4+ cells that may be relevant to a specific condition is a daunting task. Consequently, the routine isolation or characterization of CD4+ T cells bearing TCRs that respond to a specific peptide have not been available in clinical or therapeutic settings.
The tremendous TCR repertoire represented by the T cell population, is the result of somatic recombination events of the alpha (α) and beta (β), or gamma (γ) and delta (δ) TCR genes of immature T cells residing in the thymus during thymic development. (Haas et al., 1993; Hayday et al., 1995) This recombination process generates a T cell repertoire with millions of different TCR combinations that collectively bind to every possible sequence of amino acids (Haas et al., 1993; Hayday et al., 1995). This repertoire circulates through vascular and lymphatic systems in a quiescent state until a T cell encounters professional APC, such as a dendritic cell (DC), with histocompatibility complexes (HLA class II) that present a peptide that binds to its TCR with appropriate affinity (Wen et al., 1998; Sakaguchi et al., 2000; Huang et al., 2012). An immunological synapse forms between the cells and the CD4+ T cell is stimulated to proliferate, generating dozens to thousands of clones. Newly produced T cells leave the secondary lymphoid organ, circulate through the body and accumulate at sites where the antigen is present. Activated CD4+ T cells release cytokines that act on nearby cells and coordinate adaptive immune responses. Days later, after the source of the antigen has been attenuated, most of the clones are eliminated by activation-induced cell death, except for a small number of that remain as memory cells, ready to trigger a rapid response should that antigen (pathogen) reappear (Wen et al., 1998; Villadangos et al., 2005).
Many CD4+ T cells isolated from fresh whole blood will divide for one or two divisions, but additional cycles of proliferation are required for the performance of clinically useful purposes. For example, the proportion of CD4+ T cells that divide relatively robustly (e.g., more than five times) in an assay period can be used an indicator of responsiveness to a query peptide. Panels that contain different fragment of a single protein or peptide can be used to create a proliferation profile that could correlate with CD4+ T cells responses to that antigen. Information derived from those results can then be used to determine the status and possible courses of action for a variety of human conditions. However, clinical characterizations of CD4+ T cells have been limited by the availability and variability in the preparation of appropriate APCs required for the successful expansion of antigen-specific CD4+ T cell populations.
Further complicating the development of diagnostic and therapeutic methods that isolate or characterize antigen-specific CD4+ T cell populations is the heterogeneity of human HLA-DR receptors in the human population. Briefly, class II HLA complexes contain a heterodimers of HLA-DRB and HLA-DRA receptors that form a binding pocket containing a peptide. The human population contains dozens, perhaps hundreds of different HLA-DRB alleles. Therefore, a large number of HLA class II alleles in the human population, combined with millions of possible TCR combinations, have made it technically difficult to isolate antigen-specific CD4+ T cells in autoimmune disorders driven by pathogenic T cells (Villdangos et al., 2005; Huang et al., 2012), such as type I diabetes (Haskins et al., 2011; Gojanovich et al., 2012; Delong et al., 2012), rheumatoid arthritis (Nikken et al., 2011; Albani et al., 2011), and multiple sclerosis (McFarland et al., 2007; Codarri et al., 2012; Sallusto et al., 2012). Put simply, TCRs that bind to a given peptide in one person may not bind an identical peptide in another because the individuals express different HLA-DRB alleles, thereby making the development of universal probes complicated and somewhat irreproducible. However, there are only two HLA-DRA alleles, one of which is found in 98% of the population. As such, methods of assessing specific CD4+ T cell responses that utilize the widely-expressed HLA-DRA allele will be applicable to the vast majority of the human population.
Accordingly, in view of the foregoing discussion, compositions and methods are described herein for providing platforms for standardizing analyses of CD4+ T cell responses. These methods and compositions include a differentiation protocol that efficiently produces myeloid dendritic cells (DC)s from human embryonic stem cells (hESC)s that can be used to stimulate antigen-specific CD4+ T cells isolated from whole blood. In sum, clinical characterizations of CD4+ T cells have been limited by variability in the preparation of primary dendritic cells (DC), but the use of stem cell-derived DCs circumvents this problem by providing a well-characterized and functionally-modifiable population of APCs for the analysis of T cell responses to any given antigen.